In indirect enzyme-linked immunohistochemical procedures, the secondary antibody serves the pivotal role of linking the primary antibody bound to the antigen of interest in a tissue section with a tertiary complex containing an enzyme reporter molecule. Two of the most commonly applied methods are the peroxidase-anti-peroxidase (PAP) (Sternberger L. A., et al., "The Unlabelled Antibody Enzyme Method of Immunohistochemistry: Preparation and Properties of Soluble Antigen-Antibody Complex (Horseradish-Antihorseradish Peroxidase) and its use in Identification of Spirochetes," J. Histochem. Cytochem., 18:315 (1970)) and the avidin-biotin-complex (ABC) method (Hsu S. M., et al., "A Comparative Study of Peroxidase-Antiperoxidase Method and an Avidin-Biotin Complex Method for Studying Polypeptide Hormones with Radioimmunoassay Antibodies," Am. J. Pathol., 75:734 (1981)). In the latter method, biotinylated secondary antibodies are applied to tissue sections in order to link peroxidase labeled avidin-biotin complexes with primary antibodies bound to a specific antigen in situ. Simplicity, high signal amplification and low background are features of the ABC method that have prompted its widespread use as an immunohistochemical detection system.
In the PAP, ABC and other indirect immunohistochemical methods, the secondary antibody used to recognize the primary antibody could bind to endogenous immunoglobulins in murine tissues. This source of non-specific background can be eliminated from many normal tissues by in vivo perfusion with buffer to wash out the vascular compartment. However, perfusion with buffer may not wash out extravasated immunoglobulins, especially from diseased or lymphoid tissues. Thus, it would be attractive to devise an immunohistochemical method that removes the need to eliminate endogenous immunoglobulins. This would greatly facilitate efforts to study normal or diseased murine tissues and to characterize transgenic mouse models of a variety of diseases. For example, immunohistochemical studies of tumors and tumor xenografts in nude mice could be conducted using a wide variety of existing mouse monoclonal antibodies (mAb) if background staining due to the presence of endogenous mouse immunoglobulins could be eliminated in tissue sections.
Several strategies have been devised to overcome this problem. For example, an acidic-potassium permanganate solution (Johansson O., et al., "Immunohistochemical Support for Three Putative Transmitters in one Neuron: Coexistence of 5-Hydroxytryptamine Substance P- and Thyrotropin Releasing Hormone-like Immunoreactivity in Medullary Neurons Projecting to the Spinal Cord," Neuroscience, 6:1857 (1981)) has been used to remove immunoglobulins from tissue sections, but this procedure damages tissues and/or the antigens of interest. Alternatively, it is possible to biotinylate the mAbs of interest and detect them with a reporter molecule coupled to avidin (Baetge E. E., et al., "Transgenic Mice Express the Human Phenylethanolamine N-Methyltransferase Gene in Adrenal Medulla and Retina," Proc. Natl. Acad. Sci., USA, 85:3648 (1988); Haspel M. V., et al., "Generation of Tumor Cell-Reactive Human Monoclonal Antibodies using Peripheral Blood Lymphocytes from Actively Immunized Colo-Rectal Carcinoma Patients," Cancer Res., 45:3951 (1985); Imam A., et al., "A Novel Immunoperoxidase Procedure of using Human Monoclonal Antibodies for the Detection of Cellular Antigens in Tissue Sections," J. Immunol. Methods, 86:17 (19B6)). However, this strategy is impractical and costly when large panels of mAbs are used.
Another potential strategy would be to block access of the anti-mouse secondary antibodies to the endogenous mouse immunoglobulins by pre-incubating the tissue sections with a low dilution of Fab' fragments from an irrelevant species that recognize endogenous mouse immunoglobulins (Foulds S., et al., "The use of Fab' fragments in a screening method for the Detection of Human Monoclonal Antibodies," Hybridoma, 9:91 (1988); Nielsen B., et al., "A Method for the Blocking of Endogenous Immunoglobulin on Frozen Tissue Sections in the Screening of Human Hybridoma Antibody in Culture Supernatants," Hybridoma, 6:103 (1987)), but this approach requires large quantities of expensive Fab' fragments. Thus, there remains a need to improve the specificity and sensitivity of indirect immunohistochemical methods.